The ubiquitously expressed Na-H exchanger NHE1 has an established function in intracellular pH and cell volume homeostasis by catalyzing electroneutral exchange of extracellular Na + for intracellular H+. During the past funding period an ancillary function of NHEI as an anchor for actin filaments was identified. Actin anchoring by NHE1 is mediated by direct binding of ERM (ezrin, radixin, moesin) actin-binding proteins, and in fibroblasts is necessary to retain the predominant localization of NHEI at the distal margin of lamellipodia, normal cell shape, and the assembly of cortical actin filaments. The objective of the current proposal is to establish an additional ancillary function of NHEI as a scaffolding platform for assembling signaling complexes and promoting signal relay. The C-terminal cytoplasmic domain of NHE1 binds directly at least 10 functionally distinct signaling molecules that coordinately regulate NHE1 activity, restrict the localization of NHE1 in specialized membrane domains, and as now proposed, may transmit signals to diverse effector pathways. Focusing on the role of NHE1 in fibroblasts, this proposal investigates the hypothesis that NHE1 acts as a spatially-restricted scaffold to assemble signaling complexes and to promote signal relay. Studies in Aim 1 will determine how NHE1 scaffolding and localization are integrated by asking how scaffolding by NHE1 determines its localization and in turn how the localization of NHE1 in lamellipodia determines its binding to identified interacting proteins. Studies in Aim 2 will test the role of an NHE1 scaffold in the assembly of signaling complexes and in signal relay. Distinct signaling units, each having a component that binds directly to NHE1, will be investigated by asking whether binding to NHE1, the localization of NHE1 in lamellipodia, or ion translocation by NHE1 is necessary for the assembly and signal relay of the unit. Representative signaling units include phosphorylation of ezrin and Arp2/3 by the Ste20-1ike kinase NIK, ezrin binding to the GTPases Rac and Cdc42, and phosphorylation of myosin light chain by the Rho kinase ROCK. Studies in Aim 3 will define the complexity of an NHE1 scaffold by using a proteomics approach to determine the dynamic composition of an NHE1 scaffold ensemble and then will ask whether the profile of associated proteins is different when NHE 1 localization or ion translocation is impaired. Established roles for NHEI in intracellular pH homeostasis, cell proliferation, neoplastic transformation, and tumor progression underscore the importance of understanding its structural and functional regulation of diverse signaling networks.